We report a versatile analytical platform for assaying multiple analytes relying on changes in longitudinal relaxation time (T1) as a result of Fe3+/Fe2+ interconversion. The T1 of water protons in Fe3+ aqueous solution differs significantly from that of Fe2+, allowing for the development of a generally applicable T1-based assay since many redox reactions enable the interconversion between Fe2+ and Fe3+ that can result in the change of T1. Compared with conventional magnetic biosensors, this T1-based assay is free of magnetic nanoparticles (MNPs), and the stability of T1-based assay is better than conventional magnetic sensors that suffer from nonspecific adsorption and aggregation of MNPs. This T1-based assay simultaneously enables "one-step mixing" assays (such as saliva sugar) and "multiple-washing" immunoassays with good stability and sensitivity, offering a promising platform for convenient, stable, and versatile biomedical analysis.